Predicting anti-tumor efficacy of multi-functional nanomedicine on decellularized hepatocellular carcinoma-on-a-chip.

Traditional hepatocellular carcinoma-chip models lack the cell structure and microenvironments necessary for high pathophysiological correlation, leading to low accuracy in predicting drug efficacy and high production costs. This study proposed a decellularized hepatocellular carcinoma-on-a-chip model to screen anti-tumor nanomedicine. In this model, human hepatocellular carcinoma (HepG2) and human normal liver cells (L02) were co-cultured on a three-dimensional (3D) decellularized extracellular matrix (dECM) in vitro to mimic the tumor microenvironments of human hepatocellular carcinoma in vivo. Additionally, a smart nanomedicine was developed by encapsulating doxorubicin (DOX) into the ferric oxide (Fe3O4)-incorporated liposome nanovesicle (NLV/Fe+DOX). NLV/Fe+DOX selectively killed 78.59% ± 6.78% of HepG2 cells through targeted delivery and synergistic chemo-chemodynamic-photothermal therapies, while the viability of surrounding L02 cells on the chip model retained high, at over 90.0%. The drug efficacy tested using this unique chip model correlated well with the results of cellular and animal experiments. In summary, our proposed hepatocellular carcinoma-chip model is a low-cost yet accurate drug-testing platform with significant potential for drug screening.

An Optimized Decellularized Extracellular Matrix from Dental Pulp Stem Cell Sheets Promotes Axonal Regeneration by Multiple Modes in Spinal Cord Injury Rats.

In the field of tissue engineering, the extracellular matrix (ECM) is considered an important element for promoting neural regeneration after spinal cord injury (SCI). Dental pulp stem cells (DPSCs), mesenchymal stem cells that originate from the neural crest, are easy to harvest and culture in vitro, express a variety of neurotrophic factors (NTFs) and deposit a large amount of ECM, making them a good choice for stem cell- or ECM-based treatment of SCI. In the present study, decellularized extracellular matrix (dECM) derived from DPSC sheets is used for the treatment of SCI. Optimization experiments reveal that incubating DPSC sheets with 1% Triton X-100 for 5 min is the best procedure for preparing DPSC dECM. It is found that DPSC dECM promotes nerve repair and regeneration after SCI and restores hindlimb motor function in rats. Mechanistically, DPSC dECM facilitates the migration and neural differentiation of neural stem cells, as well as M2 polarization of microglia, and inhibits the formation of glial scars. This study suggests that the use of DPSC dECM is a potential strategy for the treatment of SCI.

In vitrothree-dimensional volumetric printing of vitreous body models using decellularized extracellular matrix bioink.

Hyalocytes, which are considered to originate from the monocyte/macrophage lineage, play active roles in vitreous collagen and hyaluronic acid synthesis. Obtaining a hyalocyte-compatible bioink during the 3D bioprinting of eye models is challenging. In this study, we investigated the suitability of a cartilage-decellularized extracellular matrix (dECM)-based bioink for printing a vitreous body model. Given that achieving a 3D structure and environment identical to those of the vitreous body necessitates good printability and biocompatibility, we examined the mechanical and biological properties of the developed dECM-based bioink. Furthermore, we proposed a 3D bioprinting strategy for volumetric vitreous body fabrication that supports cell viability, transparency, and self-sustainability. The construction of a 3D structure composed of bioink microfibers resulted in improved transparency and hyalocyte-like macrophage activity in volumetric vitreous mimetics, mimicking real vitreous bodies. The results indicate that our 3D structure could serve as a platform for drug testing in disease models and demonstrate that the proposed printing technology, utilizing a dECM-based bioink and volumetric vitreous body, has the potential to facilitate the development of advanced eye models for future studies on floater formation and visual disorders.

3D Bioprinted Tissue-Engineered Bone with Enhanced Mechanical Strength and Bioactivities: Accelerating Bone Defect Repair through Sequential Immunomodulatory Properties.

In this study, a new-generation tissue-engineered bone capable of temporally regulating the immune response, balancing proinflammatory and anti-inflammatory activities, and facilitating bone regeneration and repair to address the challenges of delayed healing and nonunion in large-sized bone defects, is innovatively developed. Using the innovative techniques including multiphysics-assisted combined decellularization, side-chain biochemical modification, and sterile freeze-drying, a novel photocurable extracellular matrix hydrogel, methacrylated bone-derived decellularized extracellular matrix (bdECM-MA), is synthesized. After incorporating the bdECM-MA with silicon-substituted calcium phosphate and bone marrow mesenchymal stem cells, the tissue-engineered bone is fabricated through digital light processing 3D bioprinting. This study provides in vitro confirmation that the engineered bone maintains high cellular viability while achieving MPa-level mechanical strength. Moreover, this engineered bone exhibits excellent osteogenesis, angiogenesis, and immunomodulatory functions. One of the molecular mechanisms of the immunomodulatory function involves the inhibition of the p38-MAPK pathway. A pioneering in vivo discovery is that the natural biomaterial-based tissue-engineered bone demonstrates sequential immunomodulatory properties that activate proinflammatory and anti-inflammatory responses in succession, significantly accelerating the repair of bone defects. This study provides a new research basis and an effective method for developing autogenous bone substitute materials and treating large-sized bone defects.

Gal-1-mediated cytochrome p450 activation promotes fibroblast into myofibroblast differentiation in pulmonary fibrosis.

Pulmonary fibrosis (PF) results from excessive extracellular matrix (ECM) deposition and tissue remodeling after activation of fibroblasts into myofibroblasts. Abnormally deposited fibrotic ECM, in turn, promotes fibroblast activation and accelerates loss of lung structure and function. However, the molecular mediators and exact mechanisms by which fibrotic ECM promotes fibroblast activation are unclear. In a bleomycin-induced PF mouse model, we found Galectin-1 (Gal-1) expression was significantly increased in lung tissue, and overexpression of Gal-1 plasmid-transfected fibroblasts were activated into myofibroblasts. Using the decellularization technique to prepare decellularized fibrotic ECM and constructing a 3D in vitro co-culture system with fibroblasts, we found that decellularized fibrotic ECM induced a high expression of Gal-1 and promoted the activation of fibroblasts into myofibroblasts. Therefore, Gal-1 has been identified as a pivotal mediator in PF. Further, we found that decellularized fibrotic ECM delivered mechanical signals to cells through the Gal-1-mediated FAK-Src-P130Cas mechanical signalling pathway, while the CYP450 enzymes (mainly involved in CYP1A1, CYP24A1, CYP3A4, and CYP2D6 isoforms) acted as a chemical signalling pathway to receive mechanical signals transmitted from upstream Gal-1, thereby promoting fibroblast activation. The Gal-1 inhibitor OTX008 or the CYP1A1 inhibitor 7-Hydroxyflavone prevented PF in mice and inhibited the role of fibrotic ECM in promoting fibroblast activation into myofibroblasts, preventing PF. These results reveal novel molecular mechanisms of lung fibrosis formation and identify Gal-1 and its downstream CYP1A1 as potential therapeutic targets for PF disease treatmnts.

Conditioned medium of human mesenchymal stromal/stem cells cultured on decellularized extracellular matrix promotes murine skeletal muscle repair after acute injury.

Mesenchymal stromal/stem cells (MSCs) and their secretome are known to exert beneficial effects in many pathological states. However, MSCs therapeutic properties can be reduced due to unsuitable in vitro maintenance conditions. Standard culture protocols neglect the fact that MSCs exist in vivo in the closest connection with the extracellular matrix (ECM), the complex protein network providing an instructive microenvironment. We found recently that conditioned medium from human endometrial MSCs cultured on cell-derived decellularized extracellular matrix (CM-dECM) is dramatically enriched in a number of paracrine factors such as GM-CSF, FGF-2, HGF, MMP-1, MCP-1, IL-6, IL-8, CXCL-1, -2, -5, -6 (Ushakov et al., 2024). Given that several upregulated molecules belong to myokines that are known to participate in skeletal muscle regeneration, we hypothesized that CM-dECM may promote restoration of damaged muscle tissue. Here, we found that CM-dECM injections into barium chloride-injured murine m. tibialis anterior caused myofiber hypertrophy and promoted angiogenesis. Besides, CM-dECM significantly contributed to progression of murine C2C12 myoblasts cell cycle suggesting that muscle repair in vivo may be connected with stimulation of resident myoblasts proliferation. In this study, a role for secretome of endometrial MSCs cultured on dECM in injured murine skeletal muscle regeneration was outlined first. Our findings demonstrate that culture on dECM may be considered as a novel preconditioning approach enhancing MSCs therapeutic potential.

Human Chorionic Membrane-derived Tunable Hydrogels for Vascular Tissue Engineering Strategies.

One of the foremost targets in the advancement of biomaterials to engineer vascularized tissues is not only to replicate the composition of the intended tissue but also to create thicker structures incorporating a vascular network for adequate nutrients and oxygen supply. For the first time, to the best of current knowledge, a clinically relevant biomaterial is developed, demonstrating that hydrogels made from the human decellularized extracellular matrix can exhibit robust mechanical properties (in the kPa range) and angiogenic capabilities simultaneously. These properties enable the culture and organization of human umbilical vein endothelial cells into tubular structures, maintaining their integrity for 14 days in vitro without the need for additional polymers or angiogenesis-related factors. This is achieved by repurposing the placenta chorionic membrane (CM), a medical waste with an exceptional biochemical composition, into a valuable resource for bioengineering purposes. After decellularization, the CM underwent chemical modification with methacryloyl groups, giving rise to methacrylated CM (CMMA). CMMA preserved key proteins, as well as glycosaminoglycans. The resulting hydrogels rapidly photopolymerize and have enhanced strength and customizable mechanical properties. Furthermore, they demonstrate angio-vasculogenic competence in vitro and in vivo, holding significant promise as a humanized platform for the engineering of vascularized tissues.

Fibrotic extracellular matrix impacts cardiomyocyte phenotype and function in an iPSC-derived isogenic model of cardiac fibrosis.

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-ß1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

Biomimetic Scaffolds for Regeneration of Temporomandibular Joint Disc: A Narrative Review.

Defects and dysfunctions of temporomandibular joint (TMJ) disc are responsible for the majority of TMJ diseases. Current treatments in this matter are usually short-term and only palliative, thus an alternative treatment that offers long-lasting repair is in great demand. In recent years great attempts have been made to prepare an ideal scaffold, which best resembles the native TMJ disc in characteristics such as mechanical, physical and biological properties. This narrative review focuses on developments of the recent ten years in fabrication of scaffolds using decellularized tissues, natural and synthetic biomaterials for regeneration of TMJ disc and compared their properties. PubMed and Google Scholar databases were searched using the following keywords ("TMJ" OR "temporomandibular joint" OR "TMD" OR "temporomandibular disease") AND ("scaffold" OR "hydrogels"). Randomized controlled trials, randomized clinical trials, case-controls, case reports, and animal studies were included. Comments, systematic reviews, meta-analyses, and non-English papers were excluded. The study concluded that hybrid scaffolds have exhibited favorable cell attachment and proliferation. Synthetic scaffolds have shown promise in providing better control over structural properties; however, additional processes are often required to provide biomimetic cell signaling. While there is still much to learn about the ideal scaffold for TMJ disc regeneration, both natural and synthetic scaffolds have shown promise in achieving the functional, structural, biological, and mechanical properties of a native TMJ disc.

Liver click dECM hydrogels for engineering hepatic microenvironments.

Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.

The Development of a 3D PET Fibrous Scaffold Modified with an Umbilical Cord dECM for Liver Tissue Engineering.

Organ and tissue dysfunction represents a clinically significant condition. By integrating cell biology with materials science, tissue engineering enables the reconstruction and restoration of damaged tissues or organs, offering a noninvasive repair approach. In our study, we replicated the cellular growth environment by utilizing a human umbilical cord-derived decellularized extracellular matrix (dECM) as a modifying agent for the polyethylene terephthalate (PET) polymeric fiber scaffold. This allowed us to create a dECM-coated polyester fiber-based scaffold, PET-dECM, tailored for liver tissue engineering purposes. We effectively produced a decellularized human umbilical cord-derived ECM through a combined decellularization process involving trypsin/EDTA, TritonX-100, and sodium deoxycholate. The application of the dECM coating onto the PET material was accomplished through several steps, such as ester hydrolysis, EDC/NHS-activated crosslinking, and dECM conjugation. The biological performance of the PET-dECM was validated using RG cell culture assays. Notably, the dECM coating significantly improved PET's hydrophilicity and biocompatibility, thereby aiding cell adhesion, proliferation, and functional differentiation (p < 0.05). It was further found that the hepatocyte function of HepaRG was significantly enhanced on the PET-dECM, which may be attributed to the dECM's ability to facilitate the restoration of cell polarity. The PET-dECM holds promise as an effective hepatocyte culture carrier and could potentially find application in liver tissue engineering.

Engineered Regenerative Isolated Peripheral Nerve Interface for Targeted Reinnervation.

A regenerative peripheral nerve interface (RPNI) offers a therapeutic solution for nerve injury through reconstruction of the target muscle. However, implanting a transected peripheral nerve into an autologous skeletal muscle graft in RPNI causes donor-site morbidity, highlighting the need for tissue-engineered skeletal muscle constructs. Here, an engineered regenerative isolated peripheral nerve interface (eRIPEN) is developed using 3D skeletal cell printing combined with direct electrospinning to create a nanofiber membrane envelop for host nerve implantation. In this in vivo study, after over 8 months of RPNI surgery, the eRIPEN exhibits a minimum Feret diameter of 15-20 µm with a cross-sectional area of 100-500 µm2, representing the largest distribution of myofibers. Furthermore, neuromuscular junction formation and muscle contraction with a force of ≈28 N are observed. Notably, the decreased hypersensitivity to mechanical/thermal stimuli and an improved tibial functional index from -77 to -56 are found in the eRIPEN group. The present novel concept of eRIPEN paves the way for the utilization and application of tissue-engineered constructs in RPNI, ultimately realizing neuroprosthesis control through synaptic connections.

Comparative Analysis of Decellularization Methods for the Production of Decellularized Umbilical Cord Matrix.

The importance of decellularized extracellular matrix (dECM) as a natural biomaterial in tissue engineering and regenerative medicine is rapidly growing. The core objective of the decellularization process is to eliminate cellular components while maximizing the preservation of the ECM's primary structure and components. Establishing a rapid, effective, and minimally destructive decellularization technique is essential for obtaining high-quality dECM to construct regenerative organs. This study focused on human umbilical cord tissue, designing different reagent combinations for decellularization protocols while maintaining a consistent processing time. The impact of these protocols on the decellularization efficiency of human umbilical cord tissue was evaluated. The results suggested that the composite decellularization strategy utilizing trypsin/EDTA + Triton X-100 + sodium deoxycholate was the optimal approach in this study for preparing decellularized human umbilical cord dECM. After 5 h of decellularization treatment, most cellular components were eliminated, confirmed through dsDNA quantitative detection, hematoxylin and eosin (HE) staining, and DAPI staining. Meanwhile, Masson staining, periodic acid-silver methenamine (PASM) staining, periodic acid-Schiff (PAS) staining, and immunofluorescent tissue section staining results revealed that the decellularized scaffold retained extracellular matrix components, including collagen and glycosaminoglycans (GAGs). Compared to native umbilical cord tissue, electron microscopy results demonstrated that the microstructure of the extracellular matrix was well preserved after decellularization. Furthermore, Fourier-transform infrared spectroscopy (FTIR) findings indicated that the decellularization process successfully retained the main functional group structures of extracellular matrix (ECM) components. The quantitative analysis of collagen, elastin, and GAG content validated the advantages of this decellularization process in preserving and purifying ECM components. Additionally, it was confirmed that this decellularized matrix exhibited no cytotoxicity in vitro. This study achieved short-term decellularization preparation for umbilical cord tissue through a combined decellularization strategy.

Assessment and process optimization of high throughput biofabrication of immunocompetent breast cancer model for drug screening applications.

Recent advancements in 3D cancer modeling have significantly enhanced our ability to delve into the intricacies of carcinogenesis. Despite the pharmaceutical industry's substantial investment of both capital and time in the drug screening and development pipeline, a concerning trend persists: drug candidates screened on conventional cancer models exhibit a dismal success rate in clinical trials. One pivotal factor contributing to this discrepancy is the absence of drug testing on pathophysiologically biomimetic 3D cancer models during pre-clinical stages. Unfortunately, current manual methods of 3D cancer modeling, such as spheroids and organoids, suffer from limitations in reproducibility and scalability. In our study, we have meticulously developed 3D bioprinted breast cancer model utilizing decellularized adipose tissue-based hydrogel obtained via a detergent-free decellularization method. Our innovative printing techniques allows for rapid, high-throughput fabrication of 3D cancer models in a 96-well plate format, demonstrating unmatched scalability and reproducibility. Moreover, we have conducted extensive validation, showcasing the efficacy of our platform through drug screening assays involving two potent anti-cancer drugs, 5-Fluorouracil and PRIMA-1Met. Notably, our platform facilitates effortless imaging and gene expression analysis, streamlining the evaluation process. In a bid to enhance the relevance of our cancer model, we have introduced a heterogeneous cell population into the DAT-based bioink. Through meticulous optimization and characterization, we have successfully developed a biomimetic immunocompetent breast cancer model, complete with microenvironmental cues and diverse cell populations. This breakthrough paves the way for rapid multiplex drug screening and the development of personalized cancer models, marking a paradigm shift in cancer research and pharmaceutical development.

Evaluation of the effects of decellularized extracellular matrix nanoparticles incorporation on the polyhydroxybutyrate/nano chitosan electrospun scaffold for cartilage tissue engineering.

Recent research focuses on fabricating scaffolds imitating the extracellular matrix (ECM) in texture, composition, and functionality. Moreover, specific nano-bio-particles can enhance cell differentiation. Decellularized ECM nanoparticles possess all of the mentioned properties. In this research, cartilage ECM, extracted from the cow's femur condyle, was decellularized, and ECM nanoparticles were synthesized. Finally, nanocomposite electrospun fibers containing polyhydroxybutyrate (PHB), chitosan (Cs) nanoparticles, and ECM nanoparticles were fabricated and characterized. TEM and DLS results revealed ECM nanoparticle sizes of 17.51 and 21.6 nm, respectively. Optimal performance was observed in the scaffold with 0.75 wt% ECM nanoparticles (PHB-Cs/0.75E). By adding 0.75 wt% ECM, the ultimate tensile strength and elongation at break increased by about 29 % and 21 %, respectively, while the water contact angle and crystallinity decreased by about 36° and 2 %, respectively. Uneven and rougher surfaces of the PHB-Cs/0.75E were determined by FESEM and AFM images, respectively. TEM images verified the uniform dispersion of nanoparticles within the fibers. After 70 days of degradation in PBS, the PHB-Cs/0.75E and PHB-Cs scaffolds demonstrated insignificant weight loss differences. Eventually, enhanced viability, attachment, and proliferation of the human costal chondrocytes on the PHB-Cs/0.75E scaffold, concluded from MTT, SEM, and DAPI staining, confirmed its potential for cartilage tissue engineering.

Photoacoustic processing of decellularized extracellular matrix for biofabricating living constructs.

The diverse biomolecular landscape of tissue-specific decellularized extracellular matrix (dECM) biomaterials provides a multiplicity of bioinstructive cues to target cells, rendering them highly valuable for various biomedical applications. However, the isolation of dECM biomaterials entails cumbersome xenogeneic enzymatic digestions and also additional inactivation procedures. Such, increases processing time, increments costs and introduces residues of non-naturally present proteins in dECM formulations that remain present even after inactivation. To overcome these limitations, herein we report an innovative conjugation of light and ultrasound-mediated dECM biomaterial processing for fabricating dECM biomaterials. Such approach gathers on ultrasound waves to facilitate dECM-in-liquid processing and visible light photocrosslinking of tyrosine residues naturally present in dECM biomaterials. This dual step methodology unlocked the in-air production of cell laden dECM hydrogels or programmable dECM hydrogel spherical-like beads by using superhydrophobic surfaces. These in-air produced units do not require any additional solvents and successfully supported both fibroblasts and breast cancer cells viability upon encapsulation or surface seeding. In addition, the optimized photoacoustic methodology also enabled a rapid formulation of dECM biomaterial inks with suitable features for biofabricating volumetrically defined living constructs through embedded 3D bioprinting. The biofabricated dECM hydrogel constructs supported cell adhesion, spreading and viability for 7 days. Overall, the implemented photoacoustic processing methodology of dECM biomaterials offers a rapid and universal strategy for upgrading their processing from virtually any tissue. STATEMENT OF SIGNIFICANCE: Leveraging decellularized extracellular matrix (dECM) as cell instructive biomaterials has potential to open new avenues for tissue engineering and in vitro disease modelling. The processing of dECM remains however, lengthy, costly and introduces non-naturally present proteins in the final biomaterials formulations. In this regard, here we report an innovative light and ultrasound two-step methodology that enables rapid dECM-in-liquid processing and downstream photocrosslinking of dECM hydrogel beads and 3D bioprinted constructs. Such photoacoustic based processing constitutes a universally applicable method for processing any type of tissue-derived dECM biomaterials.

Periapical lesion-derived decellularized extracellular matrix as a potential solution for regenerative endodontics.

Pulp regeneration remains a crucial target in the preservation of natural dentition. Using decellularized extracellular matrix is an appropriate approach to mimic natural microenvironment and facilitate tissue regeneration. In this study, we attempted to obtain decellularized extracellular matrix from periapical lesion (PL-dECM) and evaluate its bioactive effects. The decellularization process yielded translucent and viscous PL-dECM, meeting the standard requirements for decellularization efficiency. Proteomic sequencing revealed that the PL-dECM retained essential extracellular matrix components and numerous bioactive factors. The PL-dECM conditioned medium could enhance the proliferation and migration ability of periapical lesion-derived stem cells (PLDSCs) in a dose-dependent manner. Culturing PLDSCs on PL-dECM slices improved odontogenic/angiogenic ability compared to the type I collagen group. In vivo, the PL-dECM demonstrated a sustained supportive effect on PLDSCs and promoted odontogenic/angiogenic differentiation. Both in vitro and in vivo studies illustrated that PL-dECM served as an effective scaffold for pulp tissue engineering, providing valuable insights into PLDSCs differentiation. These findings pave avenues for the clinical application of dECM's in situ transplantation for regenerative endodontics.

Desktop-Stereolithography 3D Printing of a Decellularized Extracellular Matrix/Mesenchymal Stem Cell Exosome Bioink for Vaginal Reconstruction.

BACKGROUND: 3D-printing is widely used in regenerative medicine and is expected to achieve vaginal morphological restoration and true functional reconstruction. Mesenchymal stem cells-derived exosomes (MSCs-Exos) were applyed in the regeneration of various tissues. The current study aimed to explore the effctive of MSCs-Exos in vaginal reconstruction. METHODS: In this work, hydrogel was designed using decellularized extracellular matrix (dECM) and gelatin methacrylate (GelMA) and silk fibroin (SF). The biological scaffolds were constructed using desktop-stereolithography. The physicochemical properties of the hydrogels were evaluated; Some experiments have been conducted to evaluate exosomes' effect of promotion vaginal reconstruction and to explore the mechanism in this process. RESULTS: It was observed that the sustained release property of exosomes in the hydrogel both in vitro and in vitro.The results revealed that 3D scaffold encapsulating exosomes expressed significant effects on the vascularization and musule regeneration of the regenerative vagina tissue. Also, MSCs-Exos strongly promoted vascularization in the vaginal reconstruction of rats, which may through the PI3K/AKT signaling pathway. CONCLUSION: The use of exosome-hydrogel composites improved the epithelial regeneration of vaginal tissue, increased angiogenesis, and promoted smooth muscle tissue regeneration. 3D-printed, lumenal scaffold encapsulating exosomes might be used as a cell-free alternative treatment strategy for vaginal reconstruction.

Decellularized extracellular matrix-decorated 3D nanofiber scaffolds enhance cellular responses and tissue regeneration.

The use of decellularized extracellular matrix products in tissue regeneration is quite alluring yet practically challenging due to the limitations of its availability, harsh processing techniques, and host rejection. Scaffolds obtained by either incorporating extracellular matrix (ECM) material or coating the surface can resolve these challenges to some extent. However, these scaffolds lack the complex 3D network formed by proteins and growth factors observed in natural ECM. This study introduces an approach utilizing 3D nanofiber scaffolds decorated with dECM to enhance cellular responses and promote tissue regeneration. Notably, the dECM can be customized according to specific cellular requirements, offering a tailored environment for enhanced therapeutic outcomes. Two types of 3D expanded scaffolds, namely radially aligned scaffolds (RAS) and laterally expanded scaffolds (LES) fabricated by the gas-foaming expansion were utilized. To demonstrate the proof-of-concept, human dermal fibroblasts (HDFs) seeded on these scaffolds for up to 8 weeks, resulted in uniform and highly aligned cells which deposited ECM on the scaffolds. These cellular components were then removed from the scaffolds through decellularization (e.g., SDS treatment and freeze-thaw cycles). The dECM-decorated 3D expanded nanofiber scaffolds can direct and support cell alignment and proliferation along the underlying fibers upon recellularization. An in vitro inflammation assay indicates that dECM-decorated LES induces a lower immune response than dECM-decorated RAS. Further, subcutaneous implantation of dECM-decorated RAS and LES shows higher cell infiltration and angiogenesis within 7 and 14 days than RAS and LES without dECM decoration. Taken together, dECM-decorated 3D expanded nanofiber scaffolds hold great potential in tissue regeneration and tissue modeling. STATEMENT OF SIGNIFICANCE: Decellularized ECM scaffolds have attained widespread attention in biomedical applications due to their intricate 3D framework of proteins and growth factors. Mimicking such a complicated architecture is a clinical challenge. In this study, we developed natural ECM-decorated 3D electrospun nanofiber scaffolds with controlled alignments to mimic human tissue. Fibroblasts were cultured on these scaffolds for 8 weeks to deposit natural ECM and decellularized by either freeze-thawing or detergent to obtain decellularized ECM scaffolds. These scaffolds were tested in both in-vitro and in-vivo conditions. They displayed higher cellular attributes with lower immune response making them a good grafting tool in tissue regeneration.

Sustained delivery of NT-3 and curcumin augments microenvironment modulation effects of decellularized spinal cord matrix hydrogel for spinal cord injury repair.

Decellularized extracellular matrix hydrogel, especially that derived from spinal cord (DSCM hydrogel), has been actively considered as a functional biomaterial for remodeling the extracellular matrix of the native tissue, due to its unique characteristics in constructing pro-regenerative microenvironment for neural stem cells (NSCs). Furthermore, DSCM hydrogel can provide multiple binding domains to growth factors and drugs. Therefore, both exogenous neurotrophic factors and anti-inflammatory drugs are highly desired to be incorporated into DSCM hydrogel, which may synergistically modulate the complex microenvironment at the lesion site after spinal cord injury (SCI). Herein, neurotrophin-3 (NT-3) and curcumin (Cur) were integrated into DSCM hydrogel for SCI therapy. Due to different affinities to the DSCM hydrogel, NT-3 underwent a controlled release manner, while curcumin released explosively within the first 24 h, followed by rather sustained but slower release. The integration of both NT-3 and curcumin significantly enhanced NSCs proliferation and their neuronal differentiation. Meanwhile, the release of curcumin promoted macrophages polarization into anti-inflammatory subtypes, which further facilitated NSCs differentiation into neurons. The in situ injected DSCM + NT3 + Cur hydrogel exerted superior capability in alleviating the inflammatory responses in rat contused spinal cord. Compared to DSCM hydrogel alone, DSCM + NT3 + Cur hydrogel more significantly promoted the recruitment of NSCs and their neuronal differentiation at the lesion site. These outcomes favored functional recovery, as evidenced by the improved hind limb movement. Overall, the bioactive DSCM hydrogel can serve as a multifunctional carrier for cooperatively release of growth factors and drugs, which significantly benefits microenvironment regulation and nerve regeneration after SCI.